1. Allison J Wildin, DO Medical College of Wisconsin, Milwaukee
When ANCA is negative: Challenges in Diagnosing Granulomatosis with polyangiitis
Allison J Wildin, DO
Medical College of Wisconsin, Milwaukee

2. Overview Brief review of granulomatosis with polyangiitis
Case presentation
Literature review and discussion
Conclusion

3. Features of Granulomatosis with Polyangiitis
GPA – formerly known as Wegner’s granulomatosis
Older adults, more common in Caucasians, and equal M:F ratio
Antineutrophil cytoplasmic antibody (ANCA)-associated small to medium vessel necrotizing vasculitis
Commonly perinuclear- ANCA (p-ANCA) pattern by immunofluorescence and proteinase 3 (PR3) antibody positive by enzyme immunoassay
Granulomatous inflammation of upper and lower respiratory tracts
Necrotizing, pauci-immune glomerulonephritis
“Limited” or “Localized” forms exist – most commonly upper respiratory tract
Seo p, stone j. the antineutrophil cytoplasmic antibody-associated vasculitides. Am J Med. 2004;117:39-50.
Lutalo P, D’Cruz D. Diagnosis and classification of granulomatosis with polyangiitis (aka wegner’s granulomatosis). Journal of autoimmunity. 2014;48-49:94-98.

4. HPI
MR. J
Previously healthy 25 y/o black male with worsening bilateral pulmonary infiltrates, fever, hypoxia
Presented four months prior to his PCP complaining of dry cough and dyspnea
Negative CXR
Treated with azithromycin and given an albuterol inhaler
Subsequent multiple encounters for persistent cough and dyspnea

5. HPI
One month ago he was seen in an emergency department with fever of and nonproductive cough
CXR - right upper lobe infiltrate
Treated again with azithromycin
Four days later he was admitted with worsening symptoms
Febrile, tachycardic
CT - bilateral apical consolidations, scattered pulmonary nodules, and mediastinal and hilar lymphadenopathy
Unresponsive to standard antibiotic therapy for community acquired pneumonia
Extensive work-up including bronchoscopy with BAL, brush biopsy and infectious work-up was unrevealing
Empirically started on antifungal therapy with some improvement
Discharged 10 days later on amoxicillin and itraconazole

6. HPI
One week after discharge, he was admitted with unresolved fevers, chills, dyspnea, and cough
Febrile to 101.6, tachycardic and mildly hypoxic
CT chest- progressive worsening of bilateral upper lobe infiltrates with new nodular appearing infiltrates now involving the lower lobes
Rheumatology was consulted

7. Additional History
ROS was negative apart from fatigue, cough, fever, chills and dyspnea
MedH: Attention-deficit disorder
SurgH: None
FamH: Mother has diabetes. No known family history of autoimmune or rheumatologic disease.
Meds: amoxicillin, itraconazole, amphetamine-dextroamphetamine
Allergies: NKDA
SocH: Single and lives at home with his mother. Worked as a packer in a warehouse for the preceding 6 months, but quit 1 month ago when he got sick. Stopped smoking marijuana and tobacco products one month ago. No known occupational exposures other than dust. No recent travel or sick contacts. No pets.

8. Physical exam Vitals: T 98.6 BP 138/84 P 120 SpO2 95% on 1 L O2
Gen: Not in any acute distress
Skin: No rashes or skin lesions
HEENT: PERRL, moist oral mucosa, no lesions in nose or mouth
Cardio: Tachycardic, no murmur
Resp: Diminished lung sounds. Breathing unlabored and able to speak in complete sentences
Abd: Active BS, soft and nontender
Neuro: Strength and sensation intact without deficit
MSK: No swelling, tenderness or restricted ROM

9. Labs ESR 128 C3 193 C4 19 WBC 32.4 Hgb 10.4 CK 132 Plt 430 RF 19
ANCA, MPO, PR3 negative
ANA negative
UA without RBC or protein
WBC 32.4
Hgb 10.4
Plt 430
Creatinine 0.79
Alk Phos 179
AST 40
ALT 40
Tbili 1.1
Infectious work up including blood and respiratory cultures, coccidiodes, blastomyces, histoplasma, cryptococcal, legionella, Hepatitis panel, TB and HIV negative

10. Imaging Diffuse patchy bilateral airspace opacities
Consolidations and nodular infiltrates in the upper lobes

11. Hospital Course
Differential diagnosis was broad including infectious, inflammatory and neoplastic etiologies
VATS procedure with wedge resection of right upper and lower lobes
4 days post-operatively he developed ARDS requiring intubation and mechanical ventilation and right-sided pneumothorax requiring chest tube placement
Lung biopsy histopathology demonstrated a pattern of granulomatous inflammation, vasculitis and thrombus most consistent with GPA

12. Pathology Granulomatous inflammation of the lung
Multinucleated giant cells and lung necrosis with neutrophilic infiltration

13. pathology
Vessel demonstrating dense neutrophilic infiltration consistent with vasculitis
Magnified view of vasculitic vessel showing intraluminal irregularity

14. Treatment
Due to disease severity he was started on pulse dose steroids 1 GM solumedrol x 3 days followed by prednisone1 mg/kg with plasmapheresis and IV cyclophosphamide
Responded well to treatment and extubated a week later
Post-discharge he received pulse IV cyclophosphamide every 2 weeks for 3 months and his steroids were tapered with plan to use Rituxan as maintenance therapy

15. Case Summary
ANCA negative GPA localized to the lower respiratory tract in a young black male presenting as worsening bilateral pulmonary infiltrates A challenging diagnosis to make Atypical patient Disease limited to a single organ without other “classic” features Negative serologic studies

16. Literature Review and discussion
One other case report of ANCA-negative GPA isolated to the lower respiratory tract in a Japanese journal
53 y/o F with nodular lung lesions and negative ANCA serologies diagnosed by thorascopic lung biopsy
ANCA serologies
Role in pathogenesis
Methods of testing
Utility in diagnosis
Implications of serology for disease manifestations and course
Tomizawa, H, Enomoto K, Kurokawa H, et al. [Antineutrophil cytoplasmic antibody (ANCA) negative Limited-form granulomatosis with polyangiitis of the lung diagnosed by the thorascopic lung biopsy]. Kyobu geka. 2018; 71(9):

17. ANCA and disease Pathogenesis
ANCA are directed against proteins found in azurophile (primary) granules of polymorphonuclear cells and monocytes and can be present on the cell surface or secreted into the extracellular environment in response to inflammatory stimuli
Proteinase 3 (PR3)
Myeloperoxidase (MPO)
ANCA may increase neutrophil-endothelial interactions and activate neutrophils leading to release of reactive oxygen species, lytic enzymes and pro-inflammatory cytokines
Triggered by environmental or infectious triggers in genetically pre-disposed individuals
Gomez-peurta J, Hernandez-Rodriguez J, Lopez-soto A, et al antineutrophil cytoplasmic antibody-associated vasculitidtes and respiratory disease. Chest. 2009;136(4):
Lutalo P, D’Cruz D. Diagnosis and classification of granulomatosis with polyangiitis (aka wegner’s granulomatosis). Journal of autoimmunity. 2014;48-49:94-98.

18. ANCA testing Two commonly used assays for ANCA
Indirect Immunofluorescence Assay (IIFA)
Patterns: c-ANCA, p-ANCA, atypical ANCA
Enzyme-linked Immunosorbent Assay (ELISA)
PR3 or MPO antibodies
Newer forms of this assay including capture-ELISA, chemiluminescence enzyme immunoassay, and fluoro-enzyme immunoassay
ANCA biochip
Radice, A, Bianchi L, Sinica R. Anti-neutrophil cytoplasmic antibodies methodologic aspects and clinical significance in systemic vasculitis. Autoimmunity reviews. 2013;12:
Tateyama K, Kodama S, Kishibe K, etal. A novel strategy with combined assays for detection of anti-neutrophil cytoplasmic antibody (ANCA) in clinically anca-negative granulomatosis with polyangiitis patients. Auris Nasus Larynx. 2017;44:

19. ANCA in Diagnosis
IFA is a more sensitive marker, while ELISA is more specific
Combined use of IFA and ELISA give a reported Sensitivity of 96% and Specificity of 98.5%
Positive ANCA needs to be interpreted in the context of clinical setting and histopathology
Negative ANCA does not rule-out diagnosis
Seo p, stone j. the antineutrophil cytoplasmic antibody-associated vasculitides. Am J Med. 2004;117:39-50.
Lutalo P, D’Cruz D. Diagnosis and classification of granulomatosis with polyangiitis (aka wegner’s granulomatosis). Journal of autoimmunity. 2014;48-49:94-98.
Radice, A, Bianchi L, Sinica R. Anti-neutrophil cytoplasmic antibodies methodologic aspects and clinical significance in systemic vasculitis. Autoimmunity reviews. 2013;12:

20. Classification and diagnostic criteria
Classification criteria and definitions, but no universally accepted and tested diagnostic criteria
American College of Rheumatology 1990 classification criteria
Chapel Hill Consensus Conference criteria
European Medicines Agency (EMA) Algorithm
Only the EMA algorithm includes ANCA as a diagnostic criterion
Lutalo P, D’Cruz D. Diagnosis and classification of granulomatosis with polyangiitis (aka wegner’s granulomatosis). Journal of autoimmunity. 2014;48-49:94-98.
Watts R, Lane S, Hanslik T, et al. Development and validation of a consensus methodology for the classification of the ANCA-associated vasculitides ad polyarteritis nodosa for epidemiological studies. Ann Rheum Dis. 2007;66(2):222.

21. 1990 ACR Criteria Nasal or oral inflammation Abnormal chest radiograph
Abnormal urinary sediment
Granulomatous inflammation on biopsy
Presence of 2 or more criteria yielded a sensitivity of 88% and specificity of 92%
Criteria pre-date ANCA testing
Does not discriminate GPA from microscopic polyangiitis or mimickers of GPA
Criteria have not performed well in some clinical studies
Leavitt R, Fauci A, Bloch D, et al. The American College of rheumatology 1990 criteria fir the classification of Wegner’s granulomatosis. Arthritis Rheum. 1990;33(8):1101.
Lutalo P, D’Cruz D. Diagnosis and classification of granulomatosis with polyangiitis (aka wegner’s granulomatosis). Journal of autoimmunity. 2014;48-49:94-98.
Rao, J. Allen N, Pincus T. Limitations of the 1990 American College of Rheumatology classification criteria in the diagnosis of vasculitis. Ann Intern Med. 1998;129(5):345.

22. Chapel Hill Consensus Conference criteria
Originally proposed in 1994 and revised in 2012
Necrotizing granulomatous inflammation usually involving the upper and lower respiratory tract, and necrotizing vascultitis affecting predominately small to medium vessels (e.g. capillaries, venules, arterioles, arteries, and veins. Necrotizing glomerulonephritis is common.
Jeanette J, Falk R, Bacon P, et al revised international chapel hill consensus conference nomenclature of vasculitides. Arthritis Rheum.2013:65(1):1-11.

23. European Medicines Agency algorithm
Uses criteria for EGPA, ACR Criteria, and Chapel Hill Consensus definition in a stepwise algorithm to categorize patients
Watts R, Lane S, Hanslik T, et AL. Development and validation of a consensus methodology for the classification of the ANCA-associated vasculitides and polyarteritis nodosa for epidemiological studies. Ann Rheum Dis. 2007:66(2):222.

24. Anca IN diagnosis Use of ANCA in diagnosis is challenging
ANCA can be positive in the absence of autoimmune vasculitis
Presence of ANCA can help increase suspicion and support the diagnosis in the right clinical scenario
10-20% of patients with GPA are ANCA negative
5-30% with localized GPA are ANCA negative
One Japanese study reported approximately 50% of limited GPA defined as GPA localized to the upper respiratory tract were ANCA negative
Some of these studies did not use combined assays
Seo p, stone j. the antineutrophil cytoplasmic antibody-associated vasculitides. Am J Med. 2004;117:39-50.
Lutalo P, D’Cruz D. Diagnosis and classification of granulomatosis with polyangiitis (aka wegner’s granulomatosis). Journal of autoimmunity. 2014;48-49:94-98.
Radice, A, Bianchi L, Sinica R. Anti-neutrophil cytoplasmic antibodies methodologic aspects and clinical significance in systemic vasculitis. Autoimmunity reviews. 2013;12:
Tateyama K, Kodama S, Kishibe K, etal. A novel strategy with combined assays for detection of anti-neutrophil cytoplasmic antibody (ANCA) in clinically anca-negative granulomatosis with polyangiitis patients. Auris Nasus Larynx. 2017;44:

25. Theories about negative ANCA
Tateyama et al proposed that some patients might have ANCA that is not able to be detected by current methods or that alternate pathogenic mechanisms exist
Minor antigens and antibodies (azurocidin, bactericidal permeability increasing protein, cathepsin G, elastase, lactoferrin, and lysozyme (LAMP-2))
T-cell dependent mechanisms
Tateyama K, Kodama S, Kishibe K, etal. A novel strategy with combined assays for detection of anti-neutrophil cytoplasmic antibody (ANCA) in clinically anca-negative granulomatosis with polyangiitis patients. Auris Nasus Larynx. 2017;44:
Gomez-peurta J, Hernandez-Rodriguez J, Lopez-soto A, et al antineutrophil cytoplasmic antibody-associated vasculitidtes and respiratory disease. Chest. 2009;136(4):

26. ANCA status implications on disease course
ANCA negative disease is more likely to be “limited” or “localized,” and less severe
Patients with ANCA-negative disease may be at higher risk for relapse and progression to systemic disease
In cases of progressive disease, ANCA may subsequently become positive
Miloslavsky E, Lu N, Unizony S, et al. Myeloperoxidase-antineutrophil cytoplasmic antibody (anca)-positive and anca-negative patients with granulomatosis with polyangiitis (Wegner’s): distinct patient subsets. Arthritis Rheumatol. 2016;68:
Gomez-peurta J, Hernandez-Rodriguez J, Lopez-soto A, et al antineutrophil cytoplasmic antibody-associated vasculitidtes and respiratory disease. Chest. 2009;136(4):

27. Conclusion
Localized forms of GPA with negative ANCA can be difficult to diagnose
Negative ANCA cannot be used to definitively rule-out autoimmune vasculitis
High level of suspicion and tissue diagnosis is essential